In sample analysis instrumentation, and especially in separation systems such as liquid chromatography and capillary electrophoresis systems, smaller dimensions will generally result in improved performance characteristics and at the same time result in reduced production and analysis costs. In this regard, miniaturized separation systems provide more effective system design, result in lower overhead due to decreased instrumentation sizing and additionally enable increased speed of analysis, decreased sample and solvent consumption and the possibility of increased detection efficiency.
Accordingly, several approaches towards miniaturization for liquid phase analysis have developed in the art; the conventional approach using drawn fused-silica capillary, and an evolving approach using silicon micromachining. What is currently thought of as conventional in miniaturization technology is generally any step toward reduction in size of the analysis system.
In conventional miniaturized technology the instrumentation has not been reduced in size; rather, it is the separation compartment size which has been significantly reduced. As an example, micro-column liquid chromatography (.mu.LC) has been described wherein columns with diameters of 100-200 .mu.m are employed as compared to prior column diameters of around 4.6 mm.
Another approach towards miniaturization has been the use of capillary electrophoresis (CE) which entails a separation technique carried out in capillaries 25-100 .mu.m in diameter. CE has been demonstrated to be useful as a method for the separation of small solutes. J. Chromatogr. 218:209 (1981); Analytical Chemistry 53:1298 (1981). In contrast, polyacrylamide gel electrophoresis was originally carried out in tubes 1 mm in diameter. Both of the above described "conventional" miniaturization technologies (.mu.LC and CE) represent a first significant step toward reducing the size of the chemical portion of a liquid phase analytical system. A major drawback in the available approaches to miniaturization involves the chemical activity and chemical instability of silicon dioxide (SiO.sub.2) substrates, such as silica, quartz or glass, which are commonly used in both CE and .mu.LC systems. More particularly, silicon dioxide substrates are characterized as high energy surfaces and strongly adsorb many compounds, most notably bases. The use of silicon dioxide materials in separation systems is further restricted due to the chemical instability of those substrates, as the dissolution of SiO.sub.2 materials increases in basic conditions (at pHs greater than 7.0).
However, despite the recognized shortcomings with the chemistry of SiO.sub.2 substrates, those materials are still used in separation systems due to their desirable optical properties. In this regard, potential substitute materials which exhibit superior chemical properties compared to silicon dioxide materials are generally limited in that they are also highly adsorbing in the UV region, where detection is important.
In order to avoid some of the substantial limitations present in conventional .mu.LC and CE techniques, and in order to enable even greater reduction in separation system sizes, there has been a trend towards providing planarized systems having capillary separation microstructures. In this regard, production of miniaturized separation systems involving fabrication of microstructures in silicon by micromachining or microlithographic techniques has been described. See, e.g. Fan et al., Anal. Chem. 66(1):177-184 (1994); Manz et al., Adv. Chrom. 33:1-66 (1993); Harrison et al., Sens. Actuators, B10(2):107-116 (1993); Manz et al., Trends Anal. Chem. 10(5):144-149 (1991); and Manz et al., Sensors and Actuators B (Chemical) B1(1-6):249-255 (1990).
Recently, sample preparation technologies have been successfully reduced to miniaturized formats. Gas chromatography (Widmer et al. (1984) Int. J. Environ. Anal. Chem. 18:1), high pressure liquid chromatography (Muller et al. (1991) J. High Resolut. Chromatogr. 14: 174; Manz et al. . (1990) Sensors & Actuators B1:249; Novotny et al., eds. (1985) Microcolumn Separations: Columns, Instrumentation and Ancillary Techniques (J. Chromatogr. Library, Vol. 30); Kucera, ed. (1984) Micro-Column High Performance Liquid Chromatography, Elsevier, Amsterdam; Scott, ed. (1984) Small Bore Liquid Chromatography Columns: Their Properties and Uses, Wiley, NY; Jorgenson et al. (1983) J. Chromatogr. 255:335; Knox et al. (1979) J. Chromatogr. 186:405; Tsuda et al. (1978) Anal. Chem. 50:632) and capillary electrophoresis (Manz et al. (1992) J. Chromatogr. 593:253; Manz et al. Trends Anal. Chem. 10:144; Olefirowicz et al. (1990) Anal. Chem. 62:1872; Second Int'l Symp. High-Perf. Capillary Electrophoresis (1990) J. Chromatogr. 516; Ghowsi et al. (1990) Anal. Chem. 62:2714) have been reduced to miniaturized formats.
Capillary electrophoresis has been particularly amenable to miniaturization because the separation efficiency is proportional to the applied voltage regardless of the length of the capillary. Harrison et al. (1993) Science 261:895-897. A capillary electrophoresis device using electroosmotic fluid pumping and laser fluorescence detection has been prepared on a planar glass microstructure. Effenhauser et al. (1993) Anal. Chem. 65:2637-2642; Burggraf et al. (1994) Sensors and Actuators B20:103-110. In contrast to silicon materials (see, Harrison et al. (1993) Sensors and Actuators B10:107-116), polyimide has a very high breakdown voltage, thereby allowing the use of significantly higher voltages.
State-of-the-art chemical analysis systems for use in chemical production, environmental analysis, medical diagnostics and basic laboratory analysis must be capable of complete automation. Such a total analysis system (TAS) (Fillipini et al (1991) J. Biotechnol. 18:153; Garnet al (1989) Biotechnol. Bioeng. 34:423; Tshulena (1988) Phys. Scr. T23:293; Edmonds (1985) Trends Anal. Chem. 4:220; Stinshoff et al. (1985) Anal. Chem. 57:114R; Guibault (1983) Anal. Chem Symp. Ser. 17:637; Widmer (1983) Trends Anal. Chem. 2:8) automatically performs functions ranging from introduction of sample into the system, transport of the sample through the system, sample preparation, separation, purification and detection, including data acquisition and evaluation. Miniaturized total analysis systems have been referred to as ".mu.-TAS."
Thermal effects have been recognized to influence many of the physical and chemical parameters involved in column separation techniques. The temperature of a column device can affect, among other things, sample stability, buffer viscosity, chemical equilibria, pH and the resulting migration time for a given chemical species.
Because of the small size of the miniaturized devices incorporated in the liquid sample apparatus disclosed herein, heating and/or cooling thereof can be extremely rapid and efficient. Temperature controlling devices can be used to provide overall heating or cooling of the apparatus, localized or regional heating or cooling and temperature gradients along the length of the separation chamber.